Figure 4

Hyperamylinemia promotes amylin accumulation in the brain. A – An anti-amylin antibody (T-4157) which binds both human amylin (h-Amy) and rat amylin (r-Amy), the latter with a higher avidity (bottom panel), detects amylin in brain protein homogenate from both WT rats and HIP rats. Total (rat and human) amylin content is about 50% greater in brain tissue from HIP rats. (*P < 0.05; N = 7 animals/group). B – Matched pancreas and brain supernatant samples and plasma from the same HIP rats (N = 2) were investigated for the presence of oligomerized amylin with the T-4157 anti-amylin antibody. C – An antibody specific for human amylin demonstrates that human amylin is present only in HIP brain tissue. Western blot analysis shows that amylin aggregates with various molecular weights accumulate in the brain. D – Amylin deposition within the brain in HIP rats was demonstrated by immunohistochemistry with the T-4157 anti-amylin antibody. Amylin immunoreactivity signal in pancreatic islets (bottom) is compared to that in brain tissue (top). The incidence of amylin plaque formation is more than ten times lower in the brain than in pancreatic islets, in HIP rats. (*P < 0.05; N = 3 animals). E – Large amylin deposits, i.e. ≥ 50 μm diameter are occasionally seen in brain parenchyma (cortex). F – Diabetic (DM) HIP rats show increased amylin accumulation in the brain compared to pre-diabetic (PD) HIP rats, which can be observed in both supernatant fraction (*P < 0.05) and pellets. For quantification of the amylin immunoreactivity signal, pellets of brain homogenates from HIP rats were treated with formic acid (overnight) and guanidine hydrochloride (for two hours) to disentangle larger amylin aggregates which may not bind to the anti-amylin antibody. (N = 4 animals/group).